Atherosclerosis leads to cardiovascular disease, thus causing significant amounts of morbidity and mortality. In animal models, cannabinoid (CB)1 receptor antagonism can reduce atherosclerosis, whereas activation of CB2 receptors by agonists are atheroprotective. These findings suggest that the endocannabinoid system (ECS) has an important role in vascular homeostasis and that its perturbation may lead to clinical disease. The ECS is composed of several components, including CB receptors, ligands (2-arachidonoylglycerol, 2AG; anandamide, AEA), endocannabinoid biosynthetic enzymes, transporters, and hydrolytic enzymes that degrade 2AG and AEA thereby terminating their actions. In addition to this complexity, 2AG can be oxygenated by cyclooxygenases to yield prostaglandin glyceryl esters (PG-Gs), which can elicit pro-inflammatory activities. Although monoacylglycerol lipase (MAGL) and fatty-acid amide hydrolase (FAAH) are known to catalyze the hydrolysis of 2AG and AEA, respectively, we recently showed that carboxylesterases (CES) 1 and 2 are also efficient at hydrolyzing 2AG and PG-Gs, but not AEA. Moreover, CES1 accounted for 40-50% and 80-95% of the 2AG and PG-G hydrolysis activity in THP1 macrophages, respectively. An enzyme (Mr 31-32kDa) of un- known function was also detected by serine hydrolase activity profiling and may be responsible for the remaining glyceryl ester hydrolysis activity. Pesticides, such as organophosphates (OPs), are environmental toxicants that can be detoxified by CES; however, CES are irreversibly inhibited by the bioactive metabolites of OPs during attempted catalytic turnover. CES1 has been shown to liberate free cholesterol from neutral lipid droplets in macrophages and in the last funding cycle we showed that this function can be blocked by CES1 inhibitors. Additionally, we recently showed that human THP1 macrophage foam cells synthesize and release 2AG and PG-Gs into the culture medium and levels of these bioactive lipids are augmented by bioactive metabolites of OP pesticides. Thus, endocannabinoids may have physiological roles in the vasculature that modulate development of atherosclerosis. Since macrophages in the vessel wall express CB1 and CB2, and 2AG biosynthesis is augmented when macrophages are stimulated by external cues, it is unclear how these opposing signaling pathways are integrated to influence atherosclerosis development. Therefore, the local concentration of 2AG in the vessel wall may be an important determinant of foam cell formation and atherosclerosis. We hypothesize that the endocannabinoid tone of vessel wall macrophages is significantly perturbed by chronic exposure to oxLDL and bioactive metabolites of OP insecticides, and that elevated levels of 2AG and its COX derived metabolite, PGE2-G, modulate cholesterol metabolism in macrophages. We will test our hypothesis with the following 3 aims: SA 1. Compare the metabolism of 2AG (endocannabinoid) and PGE2-G (prostaglandin E2 glyceryl ester) in human blood-derived monocytes/macrophages and the THP-1 cell line. Identify the putative endocannabinoid hydrolytic enzyme (Mr 31-32 kDa) that we previously detected by serine hydrolase activity profiling. The working hypothesis for aim 1 is that the metabolism of lipid glyceryl esters will be similar in primary and cultured human macrophages because both cell types will likely have the same complement of endocannabinoid metabolizing enzymes, including the 31-32 kDa enzyme, which we postulate has 2AG hydrolytic activity. SA 2. Determine the mechanism by which pro-atherogenic factors activate the biosynthesis and catabolism of 2AG and PGE2-G in macrophages. The working hypothesis is that factors that contribute to atherosclerosis, such as oxidized LDL and toxicants, can modulate the metabolism of lipid glyceryl esters in macrophages. SA 3. Determine if exposure to 2AG and PGE2-G reduces cholesterol efflux from macrophages obtained from wild-type mice and CB1- and CB2-null mice. The working hypothesis is that activation of CB1 and CB2 have opposing functional effects on cholesteryl ester turnover and cholesterol efflux from macrophages. The research proposed in this application will make a major impact because it will elucidate the mechanistic details by which endogenous toxins (oxLDL) and exogenous toxicants (pesticides) can together dysregulate the endocannabinoid system in macrophages, thus enhancing foam cell development. Accordingly, combination therapies that utilize both CB1 receptor antagonists and CB2 receptor agonists could be developed in order to restore homeostasis within the vessel wall, thereby promoting human health. The studies outlined here will help to determine the feasibility of this approach. PUBLIC HEALTH RELEVANCE: The research proposed in this application will make a major impact because it will elucidate the mechanistic details by which endogenous toxins (oxLDL) and exogenous toxicants (pesticides) can together dysregulate the endocannabinoid system in macrophages, thus enhancing foam cell development. Accordingly, combination therapies that utilize both cannabinoid (CB)1 receptor antagonists and CB2 receptor agonists could be developed in order to restore homeostasis within the vessel wall, thereby promoting human health. The studies outlined here will help to determine the feasibility of this approach.